The long-term objective of the application is to identify factors contributing to the development of diabetic glomerulosclerosis and nephropathy. Diabetic nephropathy affects about 30% of all patients with diabetes mellitus and is a major reason for end-stage renal disease. Overall the proposal is based on the hypothesis that hyperglycemia leads to altered mesangial cell biology which in turn contributes to diabetic glomerular sclerosis. In order to approach this problem two potential mechanisms will be explored: (1) Glucose induced changes in cell-matrix interactions, and (2) Glucose induced changes in gene expression. Because of the intricate relation between matrix and cell, alterations in matrix will influence the biology of the cells.
For specific aim #1, the questions arise: Do glucose-induced changes in mesangial matrix and mesangial cell biology alter the subsequent expression of matrix components and cytokines by mesangial cells and/or influence the response of mesangial cells to cytokines? Mesangial cells will be used and grown on control or glucose modified matrices. The subsequent expression of matrix components and cytokines and the response to cytokines will be evaluated by biochemical, radioimmunological and molecular biology methodology. Changes in extracellular glucose concentrations can influence the expression of a number of genes, including matrix components. The mechanism(s) underlying the glucose-induced changes in gene expression remain unknown. Hyperglycemia could influence gene expression via changes in protein kinase C activity and the cellular redox state and result in changes of transcription.
In specific aim 2 the question will be addressed: Does glucose alter gene expression via changes in diacylglycerol - protein kinase C, via changes in intracellular redox potential, and via activation of specific transcription factors? Pharmacological interventions will be designed to examine protein kinase C - medicated steps and changes in redox potential. The effect of the various manipulations will be evaluated by determination of the steady- state levels of mRNA for gene products influenced by kinase C, such as the early response genes c-fos and c-jun, collagen and laminin and TGF-beta. Potential activation of the AP-1 transcription factors will be determined by gel-shift assays. CAT-reporter assays will be used to evaluate the resulting transcriptional activation. Taken together, these different approaches should provide information on the roles of glucose in the altered matrix-cell interactions and in the activation of genes by mesangial cells. The answers will not only be of interest for the development of diabetic glomerulophathy, but may also apply to diabetic tissue damage in general.
